重组人乳铁蛋白对口腔癌KB细胞增殖及凋亡影响的研究  

Recombinant human lactoferrin inhibits proliferation and induces apoptosis in oral cancer KB cells

作　　者：肖莉 张继斌 李宝霞 XIAO Li;ZHANG Jibin;LI Baoxia(Department of Stomatology,Panyu Central Hospital,Guangzhou 511400,China)

机构地区：[1]广州市番禺区中心医院口腔科,广东广州511400 [2]中山大学肿瘤防治中心,华南肿瘤国家重点实验室,广东广州510600

出　　处：《口腔医学》2018年第9期785-790,共6页Stomatology

基　　金：广东省科技计划(2013B021800049)

摘　　要：目的研究重组人乳铁蛋白(recombinate human lactoferrin,rh LF)对人口腔KB细胞增殖及凋亡的影响,为口腔癌患者提供新的有效治疗途径提供参考。方法采用CCK-8法检测不同浓度(0、12.5、25、50、100、200μg/m L)的rh LF分别处理KB细胞24、48、72 h后对细胞增殖的影响;Immunofluorescence及Western blotting方法检测rh LF处理前后DNA损伤相关蛋白γ-H2AX的表达。并分别用0、50、200μg/m L的rh LF处理KB细胞,14 d后观察其对集落克隆形成的影响;0、200μg/m L rh LF处理KB细胞48 h后,收集细胞并使用线粒体膜电位检测试剂盒(JC-1)检测rh LF处理前后线粒体膜电位的变化。Annexin VPI染色检测rh LF对口腔癌KB细胞凋亡的影响;Western blotting检测0、50、200μg/m L的rh LF处理口腔癌KB细胞48 h后Parp、Caspase-3的表达情况。结果 rh LF处理KB细胞的增殖有显著的抑制作用。Immunofluorescence结果显示rh LF处理后γ-H2AX的表达明显增加,Western blotting结果也显示rh LF处理后γ-H2AX的表达成浓度依赖性增加。rh LF抑制KB细胞集落克隆的形成;JC-1荧光检测结果表明红绿荧光的相对比例降低,这种红绿荧光的相对比例降低提示膜电位下降;0、50、200μg/m L的rh LF处理口腔癌KB细胞48 h的凋亡率为2.02%、7.60%、48.07%;同时rh LF刺激口腔癌KB细胞能诱导Parp、Caspase-3的激活。结论 rh LF能显著抑制KB细胞的增殖并且诱导其细胞凋亡,其机制与线粒体凋亡途径的激活应激有关。Objective To investigate the effects of rhLF on the proliferation and apoptosis in oral cancer KB cell line in order to a- chieve a new effective treatment for oral cancer patients. Methods KB cells were treated with different doses of rhLF （0,12.5,25,50, 100,200 μg/mL） for 24,48,72 h and cell viability was detected by CCK-8 assay. Immunofluorescence was used to evaluate the expres- sion of DNA damage associated protein ,γ-H2AX. Western blotting was performed to examine the expressions of γ-H2AX. Colony forma- tion of the ceils was observed after KB cells had been treated with rhLF at 0,200μg/mL for 14 days. JC-1 assay was used to measure the changes in mitochondrial membrane potential, Annexin V-PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the expressions of Parp and Caspase-3. Results rhLF caused significant suppression of KB cells proliferation. Im- munofluorescence showed the increase of γ-H2AX after cells were incubated with rhLF. Western blotting displayed that the expressions of γ-H2AX gradually increased in dose-dependent. Meanwhile, rhLF significantly inhibited KB cells colony formation. Exposure to rhLF （0, 50, 200 μg/mL） after 48 h resulted in 2.02%, 7.60% and 48.07% cell apoptotic rates. Exposure of the cells to increased concen- trations of rhLF gradually increased the apoptotic rate and decreased mitochondrial membrane potential, rhLF increased the expression of activated Parp, Caspase-3. Conclusion rhLF can inhibit the proliferation and induce apoptosis of KB cells, the rhLF of which may be associated with the activation of the mitochondrial apoptotic pathway.

关 键 词：口腔癌 重组人乳铁蛋白 凋亡 

分 类 号：R394.2[医药卫生—医学遗传学]